Cow’s milk protein β-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells

doi:10.1016/j.jaci.2020.05.023

Journal of Allergy and Clinical Immunology

Volume 147, Issue 1, January 2021, Pages 321-334.e4

https://doi.org/10.1016/j.jaci.2020.05.023 Get rights and content

Background

Beta-lactoglobulin (BLG) is a bovine lipocalin in milk with an innate defense function. The circumstances under which BLG is associated with tolerance of or allergy to milk are not understood.

Objective

Our aims were to assess the capacity of ligand-free apoBLG versus loaded BLG (holoBLG) to protect mice against allergy by using an iron-quercetin complex as an exemplary ligand and to study the molecular mechanisms of this protection.

Methods

Binding of iron-quercetin to BLG was modeled and confirmed by spectroscopy and docking calculations. Serum IgE binding to apoBLG and holoBLG in children allergic to milk and children tolerant of milk was assessed. Mice were intranasally treated with apoBLG versus holoBLG and analyzed immunologically after systemic challenge. Aryl hydrocarbon receptor (AhR) activation was evaluated with reporter cells and Cyp1A1 expression. Treated human PBMCs and human mast cells were assessed by fluorescence-activated cell sorting and degranulation, respectively.

Results

Modeling predicted masking of major IgE and T-cell epitopes of BLG by ligand binding. In line with this modeling, IgE binding in children allergic to milk was reduced toward holoBLG, which also impaired degranulation of mast cells. In mice, only treatments with holoBLG prevented allergic sensitization and anaphylaxis, while sustaining regulatory T cells. BLG facilitated quercetin-dependent AhR activation and, downstream of AhR, lung Cyp1A1 expression. HoloBLG shuttled iron into monocytic cells and impaired their antigen presentation.

Conclusion

The cargo of holoBLG is decisive in preventing allergy in vivo. BLG without cargo acted as an allergen in vivo and further primed human mast cells for degranulation in an antigen-independent fashion. Our data provide a mechanistic explanation why the same proteins can act either as tolerogens or as allergens.

Graphical abstract

Section snippets

Ethical approval

Volunteers donated blood after providing written informed consent. The study was approved by the ethics committee (approval no. 1972/2017) of the Medical University of Vienna and conducted in accordance with the principles of the Helsinki Declaration of 1975. Sera of patients with milk allergy (20 patients who tested positive and 20 patients who tested negative to oral cow’s milk allergen challenge) were retrospectively collected in accordance with the principles of the Helsinki Declaration of

Quercetin-iron, but not ferrioxamine complexes, are bound by BLG

Quercetin, with a complex stability constant log β of 44.2 at pH 7.4,³¹^,⁵⁹ binds strongly to ferric iron at physiologic pH. Importantly, BLG bound very strongly to FeQ2 complexes (Fig 1, A), with calculated affinities in the nM range (Fig 1, B), but not to non–catechol-based siderophores such as ferrioxamine. Complex formation with iron led to concentration-dependent quenching of quercetin fluorescence (Fig 1, C) and induced a visible color change (Fig 1, D). In spectral analysis, addition of

Discussion

Here we have revealed a previously unrecognized function of the lipocalin BLG, the major whey compound of milk, to provide immune tolerance when loaded with FeQ2 complex. Our data suggest that proper loading prevents the antigenicity of the cow's milk allergen itself, on the one hand, by masking important immune epitopes, and on the other hand, by shuttling ligands to immune cells that downtune their antigen presentation skills. HoloBLG shuttled complexed iron into antigen-presenting cells but

References (113)

T. Brick et al.
Omega-3 fatty acids contribute to the asthma-protective effect of unprocessed cow's milk
J Allergy Clin Immunol
(2016)
G. Loss et al.
The protective effect of farm milk consumption on childhood asthma and atopy: the GABRIELA study
J Allergy Clin Immunol
(2011)
M. Kaczmarski et al.
The natural history of cow's milk allergy in north-eastern Poland
Adv Med Sci
(2013)
M. Fluckinger et al.
Lipocalin-interacting-membrane-receptor (LIMR) mediates cellular internalization of beta-lactoglobulin
Biochim Biophys Acta
(2008)
D.R. Flower
Beyond the superfamily: the lipocalin receptors
Biochim Biophys Acta
(2000)
L.R. Devireddy et al.
A cell-surface receptor for lipocalin 24p3 selectively mediates apoptosis and iron uptake
Cell
(2005)
F. Roth-Walter et al.
Clinical efficacy of sublingual immunotherapy is associated with restoration of steady-state serum lipocalin 2 after SLIT: a pilot study
World Allergy Organ J
(2018)
P. Wojnar et al.
Antisense down-regulation of lipocalin-interacting membrane receptor expression inhibits cellular internalization of lipocalin-1 in human NT2 cells
J Biol Chem
(2003)
L. Chaneton et al.
Antimicrobial activity of bovine beta-lactoglobulin against mastitis-causing bacteria
J Dairy Sci
(2011)
C.D. Kanakis et al.
Milk beta-lactoglobulin complexes with tea polyphenols
Food Chem
(2011)

M. Stojadinovic et al.

Binding affinity between dietary polyphenols and beta-lactoglobulin negatively correlates with the protein susceptibility to digestion and total antioxidant activity of complexes formed

Food Chem

(2013)

H.M. Rawel et al.

Influence of a sugar moiety (rhamnosylglucoside) at 3-O position on the reactivity of quercetin with whey proteins

Int J Biol Macromol

(2003)

L. Zhang et al.

Galloyl moieties enhance the binding of (-)-epigallocatechin-3-gallate to beta-lactoglobulin: a spectroscopic analysis

Food Chem

(2017)

F. Roth-Walter et al.

Bet v 1 from birch pollen is a lipocalin-like protein acting as allergen only when devoid of iron by promoting Th2 lymphocytes

J Biol Chem

(2014)

S.F. Mirpoor et al.

Efficient delivery of quercetin after binding to beta-lactoglobulin followed by formation soft-condensed core-shell nanostructures

Food Chem

(2017)

J.M. Besle et al.

Ultraviolet-absorbing compounds in milk are related to forage polyphenols

J Dairy Sci

(2010)

E.S. Yu et al.

Regulatory mechanisms of IL-2 and IFNgamma suppression by quercetin in T helper cells

Biochem Pharmacol

(2008)

R.R. Shields-Cutler et al.

Human urinary composition controls antibacterial activity of siderocalin

J Biol Chem

(2015)

L.R. Devireddy et al.

A mammalian siderophore synthesized by an enzyme with a bacterial homolog involved in enterobactin production

Cell

(2010)

G. Kontopidis et al.

The ligand-binding site of bovine beta-lactoglobulin: evidence for a function?

J Mol Biol

(2002)

M. Carson

Ribbons. Methods Enzymol

(1997)

E. Krissinel et al.

Inference of macromolecular assemblies from crystalline state

J Mol Biol

(2007)

N. Aguilera-Montilla et al.

Aryl hydrocarbon receptor contributes to the MEK/ERK-dependent maintenance of the immature state of human dendritic cells

Blood

(2013)

E. Carrasco-Marin et al.

Iron salts and iron-containing porphyrins block presentation of protein antigens by macrophages to MHC class II-restricted T cells

Cell Immunol

(1996)

K.M. Jarvinen et al.

B-cell epitopes as a screening instrument for persistent cow's milk allergy

J Allergy Clin Immunol

(2002)

Y. Machado et al.

Fold stability during endolysosomal acidification is a key factor for allergenicity and immunogenicity of the major birch pollen allergen

J Allergy Clin Immunol

(2016)

S. Mecheri et al.

Biological effect of transferrin on mast cell mediator release during the passive cutaneous anaphylaxis reaction: a possible inhibition mechanism involving iron

Ann Inst Pasteur Immunol

(1987)

C. Esser et al.

The aryl hydrocarbon receptor in immunity

Trends Immunol

(2009)

J.A. Goettel et al.

AHR activation is protective against colitis driven by T cells in humanized mice

Cell Rep

(2016)

J. Ye et al.

The aryl hydrocarbon receptor preferentially marks and promotes gut regulatory T cells

Cell Rep

(2017)

P. Bacher et al.

Regulatory T cell specificity directs tolerance versus allergy against aeroantigens in humans

Cell

(2016)

J.A. Thorson et al.

Role of iron in T cell activation: TH1 clones differ from TH2 clones in their sensitivity to inhibition of DNA synthesis caused by IgG Mabs against the transferrin receptor and the iron chelator deferoxamine

Cell Immunol

(1991)

S. Leung et al.

Differential inhibition of inducible T cell cytokine secretion by potent iron chelators

J Biomol Screen

(2005)

G. Jordakieva et al.

Country-wide medical records infer increased allergy risk of gastric acid inhibition

Nat Commun

(2019)

M.M. Stein et al.

Innate immunity and asthma risk in amish and hutterite farm children

N Engl J Med

(2016)

M.J. Ege et al.

Exposure to environmental microorganisms and childhood asthma

N Engl J Med

(2011)

M. Waser et al.

Inverse association of farm milk consumption with asthma and allergy in rural and suburban populations across Europe

Clin Exp Allergy

(2007)

K. Jonsson et al.

Fat intake and breast milk fatty acid composition in farming and nonfarming women and allergy development in the offspring

Pediatr Res

(2016)

S. Abbring et al.

Milk processing increases the allergenicity of cow's milk-preclinical evidence supported by a human proof-of-concept provocation pilot

Clin Exp Allergy

(2019)

H.Y. Lam et al.

Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved

Clin Exp Allergy

(2008)

E. Jensen-Jarolim et al.

Structural similarities of human and mammalian lipocalins, and their function in innate immunity and allergy

Allergy

(2016)

F. Roth-Walter et al.

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches

Allergy

(2008)

A. Mansouri et al.

Plasma membrane receptor for beta-lactoglobulin and retinol-binding protein in murine hybridomas

Biofactors

(1998)

R.W. Hesselink et al.

Expression, characterization and ligand specificity of lipocalin-1 interacting membrane receptor (LIMR)

Mol Membr Biol

(2013)

Roth-Walter F, Jensen-Jarolim E, Gomez-Casado C, Diaz-Perales A, Pacios LF, Singer J. Method and means for diagnosing...

G. Bao et al.

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex

Nat Chem Biol

(2010)

M. Fluckinger et al.

Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores

Antimicrob Agents Chemother

(2004)

F. Roth-Walter et al.

The major cow milk allergen Bos d 5 manipulates T-helper cells depending on its load with siderophore-bound iron

PLoS One

(2014)

K. Hufnagl et al.

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope

Sci Rep

(2018)

J.K. Keppler et al.

Differences in binding behavior of (-)-epigallocatechin gallate to beta-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B)

J Mol Recognit

(2015)

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Citation Excerpt :
BLG is able to bind up to three different ligands simultaneously,72 including plant flavonoids and iron-flavonoid complexes, especially in the dimeric state of BLG.73 BLG binds to specific receptors on immune cells and by transporting its cargo partakes in establishing immune resilience.70,71 Therefore, BLG in combination with its ligands, so-called holo-BLG, is not an allergen, but a tolerogen.70
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The purpose of this WAO DRACMA paper is to: (i) give a comprehensive overview of milk protein allergens, (ii) to review their immunogenicity and allergenicity in the context of industrial processing, and (iii) to review the milk-related immune mechanisms triggering IgE-mediated immediate type hypersensitivity reactions, mixed reactions and non-IgE mediated hypersensitivities.
The main cow’s milk allergens – α-lactalbumin, β-lactoglobulin, serum albumin, caseins, bovine serum albumins, and others – may determine allergic reactions through a range of mechanisms. All marketed milk and milk products have undergone industrial processing that involves heating, filtration, and defatting. Milk processing results in structural changes of immunomodulatory proteins, leads to a loss of lipophilic compounds in the matrix, and hence to a higher allergenicity of industrially processed milk products. Thereby, the tolerogenic capacity of raw farm milk, associated with the whey proteins α-lactalbumin and β-lactoglobulin and their lipophilic ligands, is lost.
The spectrum of immunopathogenic mechanisms underlying cow's milk allergy (CMA) is wide. Unprocessed, fresh cow's milk, like human breast milk, contains various tolerogenic factors that are impaired by industrial processing. Further studies focusing on the immunological consequences of milk processing are warranted to understand on a molecular basis to what extent processing procedures make single milk compounds into allergens.

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: Supported by the Austrian Science Fund FWF (grant SFB F4606-B28) and in part by Biomedical International R+D GmbH, Vienna, Austria, and by Bencard Allergie GmbH, Munich, Germany. S.M.A. was supported by a grant from the Egyptian Ministry of Higher Education.

: Disclosure of potential conflict of interest: E. Jensen-Jarolim, L. F. Pacios, and F. Roth-Walter are inventors of patent EP2894478, “Method and means for diagnosing and treating allergy,” which is owned by Biomedical International R+D GmbH, Vienna, Austria. The rest of the authors declare that they have no relevant conflicts of interest.

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Mechanisms of allergy/immunologyCow’s milk protein β-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells

Background

Objective

Methods

Results

Conclusion

Graphical abstract

Section snippets

Ethical approval

Quercetin-iron, but not ferrioxamine complexes, are bound by BLG

Discussion

J Allergy Clin Immunol

J Allergy Clin Immunol

Adv Med Sci

Biochim Biophys Acta

Biochim Biophys Acta

Cell

World Allergy Organ J

J Biol Chem

J Dairy Sci

Food Chem

Food Chem

Int J Biol Macromol

Food Chem

J Biol Chem

Food Chem

J Dairy Sci

Biochem Pharmacol

J Biol Chem

Cell

J Mol Biol

Ribbons. Methods Enzymol

J Mol Biol

Blood

Cell Immunol

J Allergy Clin Immunol

J Allergy Clin Immunol

Ann Inst Pasteur Immunol

Trends Immunol

Cell Rep

Cell Rep

Cell

Cell Immunol

J Biomol Screen

Country-wide medical records infer increased allergy risk of gastric acid inhibition

Nat Commun

Innate immunity and asthma risk in amish and hutterite farm children

N Engl J Med

Exposure to environmental microorganisms and childhood asthma

N Engl J Med

Inverse association of farm milk consumption with asthma and allergy in rural and suburban populations across Europe

Clin Exp Allergy

Fat intake and breast milk fatty acid composition in farming and nonfarming women and allergy development in the offspring

Pediatr Res

Milk processing increases the allergenicity of cow's milk-preclinical evidence supported by a human proof-of-concept provocation pilot

Clin Exp Allergy

Cow's milk allergy in adults is rare but severe: both casein and whey proteins are involved

Clin Exp Allergy

Structural similarities of human and mammalian lipocalins, and their function in innate immunity and allergy

Allergy

Pasteurization of milk proteins promotes allergic sensitization by enhancing uptake through Peyer's patches

Allergy

Plasma membrane receptor for beta-lactoglobulin and retinol-binding protein in murine hybridomas

Biofactors

Expression, characterization and ligand specificity of lipocalin-1 interacting membrane receptor (LIMR)

Mol Membr Biol

Iron traffics in circulation bound to a siderocalin (Ngal)-catechol complex

Nat Chem Biol

Human tear lipocalin exhibits antimicrobial activity by scavenging microbial siderophores

Antimicrob Agents Chemother

The major cow milk allergen Bos d 5 manipulates T-helper cells depending on its load with siderophore-bound iron

PLoS One

Retinoic acid prevents immunogenicity of milk lipocalin Bos d 5 through binding to its immunodominant T-cell epitope

Sci Rep

Differences in binding behavior of (-)-epigallocatechin gallate to beta-lactoglobulin heterodimers (AB) compared to homodimers (A) and (B)

J Mol Recognit

Mechanisms of allergy/immunology
Cow’s milk protein β-lactoglobulin confers resilience against allergy by targeting complexed iron into immune cells